functional-assessment-brca1-variants-using-crispr-mediated-base

Functional Assessment of <em>BRCA1</em> variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

University of Ulsan College of Medicine

Generation of knockout mice by Cpf1-mediated gene targeting.

Senescence, defined as irreversible cell-cycle arrest, is the main driving force of aging and age-related diseases. Here, we performed high-throughput screening to identify compounds that alleviate senescence and identified the ataxia telangiectasia mutated (ATM) inhibitor KU-60019 as an effective agent. To elucidate the mechanism underlying ATM's role in senescence, we performed a yeast two-hybrid screen and found that ATM interacted with the vacuolar ATPase V subunits ATP6V1E1 and ATP6V1G1. Specifically, ATM decreased E-G dimerization through direct phosphorylation of ATP6V1G1. Attenuation of ATM activity restored the dimerization, thus consequently facilitating assembly of the V and V domains with concomitant reacidification of the lysosome. In turn, this reacidification induced the functional recovery of the lysosome/autophagy system and was coupled with mitochondrial functional recovery and metabolic reprogramming. Together, our data reveal a new mechanism through which senescence is controlled by the lysosomal-mitochondrial axis, whose function is modulated by the fine-tuning of ATM activity.

Chemical screening identifies ATM as a target for alleviating senescence.

The bacteria-derived clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are powerful tools for genome engineering. Recently, in addition to Cas protein engineering, the improvement of guide RNAs are also performed, contributing to broadening the research area of CRISPR-Cas9 systems. Here we develop a fusion guide RNA (fgRNA) that functions with both Cas9 and Cpf1 proteins to induce mutations in human cells. Furthermore, we demonstrate that fgRNAs can be used in multiplex genome editing and orthogonal genome manipulation with two types of Cas proteins. Our results show that fgRNAs can be used as a tool for performing multiple gene manipulations.

Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos.

The originally published version of this Article contained an error in the spelling of the author Da-eun Kim, which was incorrectly given as Da-Eun Kim. Furthermore, in Figure 1a, the Cas9 protein was positioned incorrectly during typesetting. These errors have now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

The CRISPR-Cas9 system is a powerful tool for genome engineering, and its programmability and simplicity have enabled various types of gene manipulation such as gene disruption and transcriptional and epigenetic perturbation. Particularly, CRISPR-based pooled libraries facilitate high-throughput screening for functional regulatory elements in the human genome. In this review, we describe recent advances in CRISPR-Cas9 technology and its use in high-throughput genetic screening. We also discuss its potential for drug target discovery and current challenges of this technique in biomedical research.

High-throughput genetic screens using CRISPR-Cas9 system.

Pooled CRISPR libraries are widely used in high-throughput screening to study various biological processes. Various pooled CRISPR libraries have been shared for CRISPR screens and useful tools have been developed to construct researcher's own libraries, however, many researchers are struggling to create their own pooled CRISPR libraries: it is a time-consuming, labor-intensive, and expensive process. In this study, we develop a simple method to customize conventional pooled CRISPR libraries using the CRISPR/Cas9 system. We show that conventional pooled CRISPR libraries can be modified by eliminating gRNAs that target positive genes, enabling the identification of unknown target genes in CRISPR screening. CRISPR/Cas9 system can be applied as a precise tool for customizing conventional pooled CRISPR libraries and will broaden the scope of high-throughput screening technology.

CRISPR/Cas-based customization of pooled CRISPR libraries.

Although mesenchymal stromal cells (MSCs) are among the most promising cell sources for cell-based therapies and regenerative medicine, the decline in their function with age due to cellular senescence limits their therapeutic applications. Unveiling the underlying mechanism of MSC senescence is therefore of substantial interest with regard to advancing MSC-based cell therapies. We here show that the induction of human umbilical cord blood-derived MSC (UCB-MSC) senescence causes the predominant upregulation of Toll-like receptor 3 (TLR3). Subsequent TLR3 activation by polyinosinic-polycytidylic acid triggers the prominent features of senescence. Using a clustered regularly interspaced short palindromic repeats/Cas9 library screening system, we identified Janus kinase 1 (JAK1) as the candidate regulatory factor for TLR3-mediated MSC senescence. A JAK1 deficiency blocked the MSC senescence phenotype upon TLR3 activation and TLR3 induction. Targeting the JAK1 pathway using chemical JAK1 inhibitors also significantly suppressed TLR3-mediated MSC senescence. Importantly, we further observed that UCB-MSC senescence is driven by a senescence-associated secretory phenotype (SASP) and that interferon-β (IFN-β) is a component of TLR3-dependent SASP, whereby its autocrine actions upregulate TLR3 and suppress cell proliferation. A JAK1 depletion significantly interrupted these effects of IFN-β, indicating that JAK1 is a signaling mediator linking IFN-β activity to TLR3 expression and the process of MSC senescence. Collectively, our findings provide new mechanistic insights into UCB-MSC senescence by revealing the role of an autocrine regulatory loop of SASP evoked by TLR3 activation.

The Upregulation of Toll-Like Receptor 3 via Autocrine IFN-β Signaling Drives the Senescence of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Through JAK1.

Genetic mutations in BRCA1, which is crucial for the process of DNA repair and maintenance of genomic integrity, are known to increase markedly the risk of breast and ovarian cancers. Clinical genetic testing has been used to identify new BRCA1 variants; however, functional assessment and determination of their pathogenicity still poses challenges for clinical management. Here, we describe that CRISPR-mediated cytosine base editor, known as BE3, can be used for the functional analysis of BRCA1 variants. We performed CRISPR-mediated base-editing screening using 745 gRNAs targeting all exons in BRCA1 to identify loss-of-function variants and identified variants whose function has heretofore remained unknown, such as c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A. Our results show that CRISPR-mediated base editor is a powerful tool for the reclassification of variants of uncertain significance (VUSs) in BRCA1.

A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants.

Stress granules (SGs) are functional messenger ribonucleoprotein aggregates, and their assembly is an important cellular process required for remodeling the signaling network to cope with extensive environmental stresses. SG formation is a stepwise process that involves the formation of a stable core followed by a less stable outer shell, and this process is often hampered by faulty regulation of protein phosphorylation. It remains unclear, however, which kinase activity is essential for SG formation. Here, we screened small molecule library of kinase inhibitors using a well-validated fluorogenic SG probe. Our screen, time-lapse microscopy, and biochemical analyses identified an ATP-mimetic SG inhibitor that selectively interferes with the fusion and growth, rather than the initial assembly, of SG core structures into the large assemblies. Thus, SGs utilize ATP-dependent chemical reactions to achieve their functional architectures.

ATP-driven reactions are required for the assembly of large stress granules.

The clinical utility of BRCA1/2 genotyping was recently extended from the selection of subjects at high risk for hereditary breast and ovary cancer to the identification of candidates for poly (ADP-ribose) polymerase (PARP) inhibitor treatment. This underscores the importance of accurate interpretation of BRCA1/2 genetic variants and of reducing the number of variants of uncertain significance (VUSs). Two recent studies by Findlay et al. and Starita et al. introduced high-throughput functional assays, and proactively analyzed variants in specific regions regardless of whether they had been previously observed. We retrospectively reviewed all BRCA1 and BRCA2 germline genetic test reports from patients with breast or ovarian cancer examined at Asan Medical Center (Seoul, Korea) between September 2011 and December 2018. Variants were assigned pathogenic or benign strong evidence codes according to the functional classification and were reclassified according to the ACMG/AMP 2015 guidelines. Among 3684 patients with available BRCA1 and BRCA2 germline genetic test reports, 429 unique variants (181 from BRCA1) were identified. Of 34 BRCA1 variants intersecting with the data reported by Findlay et al., three missense single-nucleotide variants from four patients (0.11%, 4/3684) were reclassified from VUSs to likely pathogenic variants. Four variants scored as functional were reclassified into benign or likely benign variants. Three variants that overlapped with the data reported by Starita et al. could not be reclassified. In conclusion, proactive high-throughput functional study data are useful for the reclassification of clinically observed VUSs. Integrating additional evidence, including functional assay results, may help reduce the number of VUSs.

Impact of proactive high-throughput functional assay data on BRCA1 variant interpretation in 3684 patients with breast or ovarian cancer.

Protein import across the endoplasmic reticulum membrane is physiologically regulated in a substrate-selective manner to ensure the protection of stressed ER from the overload of misfolded proteins. However, it is poorly understood how different types of substrates are accurately distinguished and disqualified during translocational regulation. In this study, we found poorly assembled translocon-associated protein (TRAP) complexes in stressed ER. Immunoaffinity purification identified calnexin in the TRAP complex in which poor assembly inhibited membrane insertion of the prion protein (PrP) in a transmembrane sequence-selective manner, through translocational regulation. This reaction was induced selectively by redox perturbation, rather than calcium depletion, in the ER. The liberation of ERp57 from calnexin appeared to be the reason for the redox sensitivity. Stress-independent disruption of the TRAP complex prevented a pathogenic transmembrane form of PrP (ctmPrP) from accumulating in the ER. This study uncovered a previously unappreciated role for calnexin in assisting the redox-sensitive function of the TRAP complex and provided insights into the ER stress-induced reassembly of translocon auxiliary components as a key mechanism by which protein translocation acquires substrate selectivity.

Protein Translocation Acquires Substrate Selectivity Through ER Stress-Induced Reassembly of Translocon Auxiliary Components.

Rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9-mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. We addressed the clinical utility of this CRISPR/dCas9-mediated biosensor in tick-borne illnesses including scrub typhus (ST) and severe fever with thrombocytopenia syndrome (SFTS), whose clinical presentations are too similar to be easily differentiated. By using CRISPR/dCas9-mediated biosensor, we achieved single molecule sensitivity for the detection of ST (0.54 aM) and SFTS (0.63 aM); this detection sensitivity is 100 times more sensitive than that of RT-PCR assay. Finally, CRISPR/dCas9-mediated biosensor was able to clearly distinguish between ST and SFTS in serum samples within 20 min. We believe that CRISPR/dCas9-mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications.

CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.

Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.

CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.

Yongsub Kim

Department of Biomedical Sciences,
Asan Medical Institute of Convergence Science and Technology,
Asan Medical Center,
Stem Cell Immunomodulation Research Center,
Department of Biomedical Sciences, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center

University of Ulsan College of Medicine

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors